Papillomaviruses (PV) have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa. Tens of millions of women suffer from human papilloma virus (HPV) infection of the genital tract. Approximately 500,000 women worldwide develop cancer of the cervix each year and it is the second most common cause of cancer deaths in women worldwide. Approximately 90-95% of all cervical cancers may be linked to HPV.
In addition to causing severe cancerous conditions, Papillomaviruses more commonly induce benign, dysplastic and malignant hyperproliferations of skin and mucosal epithelium (see, for example, Mansur and Androphy, (1993) Biochem Biophys Acta 1155:323-345; Shah and Howley (1996) Fields Virology, 3rd Ed., 2077-2110; and Howley (1996) Fields Virology 3rd Ed., 2045-2076, for reviews of the molecular, cellular, and clinical aspects of the papillomaviruses). For example, a wide variety of warts are found on human skin and are caused by the human papillomavirus (HPV). Examples of such warts include common warts (verruca vulgaris), plantar warts, palmar warts, planar warts (verruca plana), mosaic warts, and venereal warts (condyloma accuminatum). These skin growths are unsightly, irritating, and potentially carcinogenic, and their removal is desirable.
Over 70 different HPV types have been identified, and these different papillomavirus types are known to cause distinct diseases, c.f., zur Hausen, (1996) Biophysica Acta 1288:55-78; Pfister, (1987) Adv. Cancer Res., 48:113-147; and Syrjanen, (1984) Obstet. Gynecol. Survey 39:252-265. The HPVs that cause anogenital warts can be further classified either high risk (such as HPV type 16 [HPV-16] and HPV-18) or low risk (or HPV-6 and HPV-11) on the basis of the clinical lesions with which they are associated and the relative propensity for these lesions to progress to cancer. For example, HPV types 1 and 2 cause common warts, and types 6 and 11 cause warts of the external genitalia, anus and cervix. HPV""s of the high risk classification can be identified in the majority of cervical cancers, e.g., approximately 90% of human cervical cancers harbor the DNA of a high-risk HPV. Types 16, 18, 31 and 33 are particularly common; HPV-16 is present in about 50 percent of all cervical cancers.
The genetic organization of the papillomaviruses are defined by ten open reading frames (ORFs) which are located on one strand of the viral DNA. These 10 genes are classified as early (E) and late (L) genes, depending on the time at which they are expressed in the viral life cycle, as shown in FIG. 1. Several of the papillomavirus early genes code for regulatory proteins that are of particular interest. The high-risk HPV encoded E6 protein, for example, causes p53 degradation and the E7 protein interacts with Rb and other pocket proteins (Howley et al., Fields Virology, Third Ed., Philadelphia: 1996). Notably, the E1 and E2 regulatory proteins play important roles in papillomavirus transcriptional regulation and viral DNA replication.
The biological life cycle of the papillomaviruses appears to differ from most other viral pathogens. These viruses are believed to infect the basal or stem cells of the squamous epithelium. Rather than proceeding to a lytic infection in which viral replication kills the cell, viral DNA transcription and replication are maintained at very low levels until the infected epithelial cell migrates into the upper strata of the squamous epithelium. There, presumably in response to differentiation-specific signals, the progression of viral transcription changes, viral DNA synthesis begins and infectious virions assemble. In this type of replication, hundreds of copies of the genomes are produced per cell, and these are packaged into progeny virions. High levels of E1 and E2 are observed in cells undergoing papillomavirus vegetative replication.
The papillomavirus E1 protein is the most conserved papillomavirus protein. The HPV-16 E1 protein is 649 amino acids long and has a molecular weight of 68-kd. The E1 protein is the only papillamovirus encoded DNA replication enzyme. All other replication enzymes, e.g., polymerases, primases, etc. are provided by the infected host cell. The E1 protein is a nuclear phosphoprotein and ATP-dependent DNA helicase that binds to the origin of DNA replication (ori), thus initiating viral plasmid replication (Howley 1996). The minimal DNA binding domain of the E1 proteins is found in the amino terminus. The HPV-16 E1 carboxy-terminus contains a domain that is necessary and sufficient for interaction with the E2 protein. This domain also includes an ATP binding pocket similar to that found in SV40 large T antigen (LgT).
Interestingly, the papillomavirus E1 protein shares many structural and functional similarities with the large T antigen (LgT) of simian virus (SV40), the replication protein of that virus. Both E1 and LgT bind a region of dyad symmetry within the DNA replication origin and have ATP-dependent DNA helicase activity that can catalyze the unwinding of ori-containing double-stranded DNA templates (Smelkova and Borowiec, Journal of Virology, 1997, 71, 8766; Chen et al., Journal of Virology, 1997, 72, 2567). One important difference between the two proteins is that LgT is the only viral protein required for initiation of SV40 replication (Bullock et al., Critical Reviews in Biochemistry and Molecular Biology, 1997, 32, 503), whereas E1 is efficiently recruited to the viral DNA replication origin, only through interaction with the virus encoded E2 protein (see below).
The papillomavirus E2 protein is a critical regulator of both viral DNA replication and gene expression. HPV-16 E2 is 365 amino acids long and has a molecular weight of 38-kD. E2 proteins are composed of two well-conserved functional domains, as shown in FIG. 2. The E2 carboxy-terminus includes a DNA binding domain that binds as a dimer to the ACCN6GGT recognition sequence (Andropy et al., Nature, 1987, 325, 70). The E2 amino-terminus features a transcriptional activation domain that regulates viral gene expression and interacts with components of the host cell apparatus. The E2 N-terminus also interacts with the E1 protein. These amino-terminal and the carboxy-terminal domains are connected by a hinge region that is dispensable for both replication and transcriptional activation.
Genetic studies have revealed that stable BPV-1 plasmid replication requires the expression of the viral E1 and E2 genes (DelVecchio et al., Journal of Virology, 1992, 66, 5949; Di Maio et al., EMBO, 1988, 7, 1197; Sarver et al., Journal of Virology, 1984, 52, 377). Though E1 and E2 are both essential factors for stable DNA replication, it appears that only E1 is directly involved in plasmid replication (DiMaio and Settlement 1986, Ustav et al. 1991). However, extensive research over the past ten years has been conducted to elucidate the role of E2 in DNA replication, and the results of these numerous studies suggest that (1) HPV-16 E2 transactivation functions are independent of and separable from its E1 interaction (i.e., replication activity), (2) E1 binding is necessary for E2 stimulation of DNA replication. E2, by binding to ACC 6GT recognition sequences that flank the viral DNA replication origin, recruits E1 to the origin. In this way, E2 effects initiation of the formation of the viral replication complex.
Thus, since the formation of an E1-E2 complex is necessary for stimulation of DNA replication, moieties that can block the formation of this complex could serve to inhibit viral DNA replication and thus the proliferation of harmful conditions as described above.
Towards this end, European patent application 302,758 refers to the use of modified forms of E2 protein that bind to, and block, E2 binding sites on papillomavirus DNA without resulting in trans-activation. That application also refers to repression of E2 activation through the use of DNA fragments that mimic E2 binding sites, and thus bind with E2 trans-activators, making them unavailable for binding to E2 sites on the viral DNA.
Additionally, U.S. Pat. No. 5,219,990 describes the use of E2 trans-activation repressors which interfere with normal functioning of the native full-length E2 transcriptional activation protein of the papillomavirus. However, the E2 trans-activation repressors of the ""990 patent are proteins that dimerize with the full-length native E2 protein to form inactive heterodimers, thus interfering with the formation of active homodimers comprising full-length native E2 polypeptides and thereby repressing papillomavirus transcription and replication. The E2 trans-activation repressors are described as fragments of the E2 polypeptide in which the dimerization function has been separated from its DNA binding function, e.g., the E2 trans-activation repressors includes at least the dimerization region, but less than the DNA binding domain, of the E2 polypeptide.
Although these references are directed towards the blocking of complex formation, each of these references describes the use of large biological molecules, namely proteins, to achieve this goal. Clearly, there remains a need for the development of simpler, preferably cell-permeable, small molecule therapeutics capable of treating a papillomavirus-induced condition, lessening the severity of such condition, or preventing the occurrence of such condition.
One aspect of the present invention provides a method of treating, e.g., lessening the severity or preventing the occurrence of a papillomavirus-induced condition, such as a papillomavirus-induced lesion. In general, the subject method comprises administering to an animal, e.g. a human, infected with a papillomavirus a pharmaceutical preparation comprising a therapeutically effective amount of a small organic molecule which can inhibit E1-E2 interaction. In preferred embodiments, the inhibitor has a molecular weight of less than 10,000 amu, more preferably less than 7500 amu, 5000 amu, and even more preferably less than 3000 amu. For instance, the E2 inhibitor can be either (i) an E2 peptide or peptidomimetic, preferably corresponding in length to a 3-25 mer, e.g., in certain embodiments, containing a core sequence of R-X(4)-E-X(5)-X(6)-X(7) and in other embodiments, including the core sequence W-X(1)-X(2)-X(3)-R-X(4)-E-X(5)-X(6)-X(7)-X(8)-X(9)-X(10)-A-X(11), or (ii) a gene construct for expressing the E2 peptide. The E2 peptide, peptidomimetic or gene construct is formulated in the pharmaceutical preparation for delivery into PV-infected cells of the animal.
In preferred embodiments, the subject method is used to treat a human who is infected with a human papillomavirus (HPV), particularly a high risk HPV such as HPV-16, HPV-18, HPV-31 and HPV-33. In other preferred embodiments, treatment of low risk HPV conditions, e.g., particular topical treatment of cutaneous or mucosal low risk HPV lesions, is also contemplated.
The subject method can be used to inhibit pathological progression of papillomavirus infection, such as preventing or reversing the formation of warts, e.g. Plantar warts (verruca plantaris), common warts (verruca plana), Butcher""s common warts, flat warts, genital warts (condyloma acuminatum), or epidermodysplasia verruciformis; as well as treating papillomavirus-infected cells which have become, or are at risk of becoming, transformed and/or immortalized, e.g. cancerous, e.g. a laryngeal papilloma, a focal epithelial, a cervical carcinoma, or as an adjunct to chemotherapy, radiation, surgical or other therapies for eliminating residual infected or pre-cancerous cells.
Yet another aspect of the invention relates to a pharmaceutical preparation comprising a therapeutically effective amount of an E2 peptidomimetic, formulated in the pharmaceutical preparation for delivery into PV-infected cells of an animal. In preferred embodiments, the polypeptide is formulated as a liposome.
As will be appreciated by one of ordinary skill in the art, the compositions and preparations described herein can also be utilized serially or in combination with conventional therapeutic agents or regimens including, but not limited to, salicylic acid, podophyllotoxin, retinoic acid, surgery, laser therapy, radiation, and cryotherapy.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained filly in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).